473
Restorative Neurology and Neuroscience 32 (2014) 473–482 DOI 10.3233/RNN-130363 IOS Press
Bone marrow-derived mononuclear cell transplantation in spinal cord injury patients by lumbar puncture Yoshihisa Suzukia,∗ , Namiko Ishikawaa , Kaoru Omaeb , Tatsuya Hiraia , Katsunori Ohnishic , Norihiko Nakanod , Hidetaka Nishidae , Toshio Nakatanif , Masanori Fukushimab and Chizuka Ided a Department
of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan b Translational Research Informatics Center, Kobe, Japan c Ikesucho-clinic, Kyoto, Japan d Institute of Regeneration and Rehabilitation, Faculty of Nursing and Rehabilitation, Aino University, Osaka, Japan e Nakayama Veterinary Hospital, Nara, Japan f Emergency and Critical Care Center, Kansai Medical University, Moriguchi, Osaka, Japan
Abstract. Purpose: This study was conducted to assess the safety and feasibility of intrathecal transplantation of autologous bone marrowderived mononuclear cells for the treatment of patients with spinal cord injury. Methods: Ten patients were included in the study. Approximately 120 ml of bone marrow aspirate was obtained from bilateral iliac bone of patients with spinal cord injury. Isolation of mononuclear cells was performed using Ficoll density-gradient centrifugation. Bone marrow mononuclear cells were transplanted into cerebrospinal fluid by lumbar puncture. Functional tests were performed prior to the cell transplantation and six months after cell transplantation. The patients were carefully observed for up to six months. Results: In 5 patients with AIS A prior to cell transplantation, 1 patient converted to AIS B six months after cell transplantation. In 5 patients with AIS B, 1 patient converted to AIS D and 2 patients to AIS C. MRI did not show any complication. Two patients showed slight anemia after aspiration of bone-marrow cells, which returned to normal level within a several weeks. Conclusion: The results of this study suggest that this method may be safe and feasible. Keywords: Central nervous system regeneration, spinal cord injury treatment, bone marrow mononuclear cell, clinical study, cell transplantation
1. Introduction Spinal cord injuries (SCIs) due to motorcycle accidents and falls occur frequently in young people in ∗ Corresponding author: Yoshihisa Suzuki, MD, PhD, Department of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8507, Japan. Tel.: +81 6 6312 1221; Fax: +81 6 6361 0588; E-mail: [email protected].
their 20 s. Owing to advances in medicine, the number of deaths due to pneumonia, urinary tract infection, or decubitus ulcers has decreased, and patients with spinal cord injury are now able to live longer. However, treatment for paralysis due to spinal cord injury has not been realized and patients who suffer a spinal cord injury at a young age must deal with incurable quadriplegia, paraplegia, pain, and bladder and rectal disturbance for the rest of their lives.
0922-6028/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
474
Y. Suzuki et al. / Mononuclear cell transplantation for treatment of spinal cord injury
Since the 1990s, studies involving animal experiments have reported that functional recovery was achieved by transplanting neural stem cells, embryonic stem (ES) cells, activated macrophages, olfactory ensheathing cells, Schwann cells, umbilical cord blood cells, and bone marrow-derived cells to the site of spinal cord injury. Furthermore, a number of clinical trials have been conducted using these cell types in recent years (Chernykh et al., 2007; Deda et al., 2009; Harrop et al., 2012; Kumar et al., 2009; Liu et al., 2013; Seberi et al., 2008). In 2010, a clinical trial using ES cells was launched by Geron Corporation in the United States (Bretzner et al., 2011). In 2011, Geron presented the safety data from four patients, and reported that the cells did not cause any serious problems. However, Geron announced it was halting this clinical trial to focus on other areas of research. We started basic research around this time (Kataoka et al., 2004; Suzuki et al., 1999) and confirmed that neural stem cells derived from the hippocampus engrafted well when transplanted directly or through cerebrospinal fluid to the site of damage in rats with spinal cord crush injuries (Wu et al., 2001, 2002). However, the development of a clinical application proved challenging because it was difficult to control the stem cells, which have a strong capacity for proliferation leading to tumor formation (Amariglio et al., 2009; Bai et al., 2003). Therefore, autologous bone marrow-derived cells were used to perform the experiment in rats. Administration of bone marrow-derived cells, stromal cells, or mononuclear cells to the cerebrospinal fluid at the 4th ventricle was examined. In the transplantation group, a profound improvement in locomotion was observed, as was a histological reduction in the severity of spinal cord injury, including cavitation (Ide et al., 2010; Nakano et al., 2010; Ohta et al., 2004; Wu et al., 2003; Yoshihara et al., 2007). On the basis of results in rats, clinical trials were initiated at veterinary hospitals in dogs with spinal cord injuries, resulting in an observed recovery from paralysis without any adverse events associated with the cell transplantation (Nishida et al., 2011; Tamura et al., 2012). Next, we conducted a human clinical trial entitled, “Investigation of spinal cord regeneration therapy by cultured autologous bone marrow stromal cell transplantation for acute spinal cord injury, Phase I/II clinical trial.” No adverse events associated with cell transplantation were observed (Saito et al., 2008, 2012). However, the efficacy of bone marrow stromal cell infusion can obviously not be evaluated solely
on the basis of the results of 5 cases. To confirm the efficacy of the treatment, it is necessary to conduct clinical trials with a larger number of patients, and a low-cost and convenient method is required. However, bone marrow stromal cells must be cultured in a cell processing center for use, and the operation and maintenance of such a center incur a cost. This was found to be a bottleneck for a multicenter trial. Therefore, it was decided to conduct a clinical trial using autologous bone marrow mononuclear cells (BMMCs) that do not require cell culture manipulation, but are expected to have equal efficacy (Samdani et al., 2009). A phase I/II trial was conducted to assess the safety and feasibility after obtaining approval from our hospital’s Ethics Committee and the Committee on Guidelines for Stem Cell Research of the Ministry of Health, Labour and Welfare, Japan. As the patients participating in the study expected some positive effect, even if only a slight one, the Ethics Committee did not approve the establishment of a control group in which patients would have received normal saline solution injection instead of cells. 2. Materials and methods 2.1. Patient selection Patients who met all of the inclusion criteria and none of the exclusion criteria were considered eligible (Table 1). 2.2. Procedure for collection of bone marrow aspirate After local anesthesia was given, if needed, a syringe was used to collect bone marrow aspirate by inserting a bone marrow needle into the posterior iliac crest. In terms of the insertion sites on the skin, the needle was inserted into the ilium at two to three sites slightly away from each other. For each iliac insertion site, 4 mL of bone marrow aspirate was collected two to three times by changing the depth of insertion. From several sites on the left and right ilium, approximately 4 mL was collected each time; a total of approximately 120 mL was collected in the end. As bone marrow aspirate will turn into a gel if it is aspirated alone, 5 mL injection syringes, which had aspirated 1 mL of saline solution containing heparin beforehand, were used. Following this, mononuclear cells were isolated in an operating
Y. Suzuki et al. / Mononuclear cell transplantation for treatment of spinal cord injury
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Table 1 Inclusion and exclusion criteria Inclusion criteria (1) Patients with spinal cord injuries classified as A–C on the ASIA impairment scale (2) Patients injured 3 weeks to 1 year previously (3) Patients with partial spinal cord injury demonstrated by diagnostic imaging (4) Patients aged between 20 and 60 years at the acquisition of informed consent (5) Patients who submitted written informed consent by themselves Exclusion criteria (1) Patients with a completely transected spinal cord (2) Patients with central spinal cord injury (3) Patients with organ failure with a SOFA score of 3 points or higher (4) Patients in whom hepatitis B, hepatitis C, human immunodeficiency virus infection, adult T-cell leukemia, or parvovirus B19 infection could not be ruled out (5) Patients with malignant tumor or a history of malignant tumor within 5 years (6) Patients with one of the following diseases/disorders: · Myeloproliferative disorder or myelodysplastic syndrome · Autoimmune disease · Spinal stenosis · Limb paralysis due to central nervous system disorder not attributed to spinal cord injury · Poorly controlled psychiatric disorder (7) Patients who were participating in other clinical trials or who completed participation within 6 months (8) Patients who were pregnant or possibly pregnant (9) Other patients who were judged to be ineligible by the investigators
Isolation, washing, and preparation of bone marrow mononuclear cells were performed on a clean bench in an operating room. The bone marrow aspirate was diluted with saline combined with human serum albumin and ACD-A solution (Terumo, Tokyo, Japan). Mononuclear cells were isolated by specific gravity centrifugation using Ficoll-Paque PREMIUM 1.073 (GE Healthcare, NY, USA). A part of the supernatant was used for a mycoplasma test, an endotoxin test, and a sterility test, and the rest was discarded. Cells were suspended in 2 mL of saline. Using some of the cells suspended in saline, hematological measurements, including cell count and cell surface markers, were performed. The survival rate of cells in the preliminary experiment was 99% or more when stored at room temperature for 12 hours; therefore, the cells were stored at room temperature until injection.
Japanese Pharmacopoeia, the notification by the Ministry of Health, Labor and Welfare titled “Ensuring the quality and safety of pharmaceuticals manufactured with components derived from human or animal as raw material” (Notification No. 1314 of the PFSB issued on Dec 26, 2000) and the minutes of the “Advisory Committee Regarding Clinical Trials Using Human Stem Cells, Council for Science and Technology, the Health Science Council,” and others. The mycoplasma test was performed using a PCR assay to examine the presence or absence of mycoplasma infection in the cells according to the 15th revised edition of Japanese Pharmacopoeia. The amount of endotoxin was measured in the cells according to the 16th revised edition of Japanese Pharmacopoeia. Measurement was performed using the supernatant removed after cell centrifugation in the procedure for the isolation of bone marrow mononuclear cells. Sterility test was performed by aerobic and anaerobic culture of the supernatant to examine the presence or absence of microorganism growth.
2.4. Quality control
2.5. Cell transplantation
For the purpose of quality control, the following tests were performed for the composition of the autologous bone marrow mononuclear cells for each subject. Quality control items were established with reference to the
The investigators confirmed that the level of endotoxin from the endotoxin test was
Restorative Neurology and Neuroscience 32 (2014) 473–482 DOI 10.3233/RNN-130363 IOS Press
Bone marrow-derived mononuclear cell transplantation in spinal cord injury patients by lumbar puncture Yoshihisa Suzukia,∗ , Namiko Ishikawaa , Kaoru Omaeb , Tatsuya Hiraia , Katsunori Ohnishic , Norihiko Nakanod , Hidetaka Nishidae , Toshio Nakatanif , Masanori Fukushimab and Chizuka Ided a Department
of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan b Translational Research Informatics Center, Kobe, Japan c Ikesucho-clinic, Kyoto, Japan d Institute of Regeneration and Rehabilitation, Faculty of Nursing and Rehabilitation, Aino University, Osaka, Japan e Nakayama Veterinary Hospital, Nara, Japan f Emergency and Critical Care Center, Kansai Medical University, Moriguchi, Osaka, Japan
Abstract. Purpose: This study was conducted to assess the safety and feasibility of intrathecal transplantation of autologous bone marrowderived mononuclear cells for the treatment of patients with spinal cord injury. Methods: Ten patients were included in the study. Approximately 120 ml of bone marrow aspirate was obtained from bilateral iliac bone of patients with spinal cord injury. Isolation of mononuclear cells was performed using Ficoll density-gradient centrifugation. Bone marrow mononuclear cells were transplanted into cerebrospinal fluid by lumbar puncture. Functional tests were performed prior to the cell transplantation and six months after cell transplantation. The patients were carefully observed for up to six months. Results: In 5 patients with AIS A prior to cell transplantation, 1 patient converted to AIS B six months after cell transplantation. In 5 patients with AIS B, 1 patient converted to AIS D and 2 patients to AIS C. MRI did not show any complication. Two patients showed slight anemia after aspiration of bone-marrow cells, which returned to normal level within a several weeks. Conclusion: The results of this study suggest that this method may be safe and feasible. Keywords: Central nervous system regeneration, spinal cord injury treatment, bone marrow mononuclear cell, clinical study, cell transplantation
1. Introduction Spinal cord injuries (SCIs) due to motorcycle accidents and falls occur frequently in young people in ∗ Corresponding author: Yoshihisa Suzuki, MD, PhD, Department of Plastic and Reconstructive Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8507, Japan. Tel.: +81 6 6312 1221; Fax: +81 6 6361 0588; E-mail: [email protected].
their 20 s. Owing to advances in medicine, the number of deaths due to pneumonia, urinary tract infection, or decubitus ulcers has decreased, and patients with spinal cord injury are now able to live longer. However, treatment for paralysis due to spinal cord injury has not been realized and patients who suffer a spinal cord injury at a young age must deal with incurable quadriplegia, paraplegia, pain, and bladder and rectal disturbance for the rest of their lives.
0922-6028/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
474
Y. Suzuki et al. / Mononuclear cell transplantation for treatment of spinal cord injury
Since the 1990s, studies involving animal experiments have reported that functional recovery was achieved by transplanting neural stem cells, embryonic stem (ES) cells, activated macrophages, olfactory ensheathing cells, Schwann cells, umbilical cord blood cells, and bone marrow-derived cells to the site of spinal cord injury. Furthermore, a number of clinical trials have been conducted using these cell types in recent years (Chernykh et al., 2007; Deda et al., 2009; Harrop et al., 2012; Kumar et al., 2009; Liu et al., 2013; Seberi et al., 2008). In 2010, a clinical trial using ES cells was launched by Geron Corporation in the United States (Bretzner et al., 2011). In 2011, Geron presented the safety data from four patients, and reported that the cells did not cause any serious problems. However, Geron announced it was halting this clinical trial to focus on other areas of research. We started basic research around this time (Kataoka et al., 2004; Suzuki et al., 1999) and confirmed that neural stem cells derived from the hippocampus engrafted well when transplanted directly or through cerebrospinal fluid to the site of damage in rats with spinal cord crush injuries (Wu et al., 2001, 2002). However, the development of a clinical application proved challenging because it was difficult to control the stem cells, which have a strong capacity for proliferation leading to tumor formation (Amariglio et al., 2009; Bai et al., 2003). Therefore, autologous bone marrow-derived cells were used to perform the experiment in rats. Administration of bone marrow-derived cells, stromal cells, or mononuclear cells to the cerebrospinal fluid at the 4th ventricle was examined. In the transplantation group, a profound improvement in locomotion was observed, as was a histological reduction in the severity of spinal cord injury, including cavitation (Ide et al., 2010; Nakano et al., 2010; Ohta et al., 2004; Wu et al., 2003; Yoshihara et al., 2007). On the basis of results in rats, clinical trials were initiated at veterinary hospitals in dogs with spinal cord injuries, resulting in an observed recovery from paralysis without any adverse events associated with the cell transplantation (Nishida et al., 2011; Tamura et al., 2012). Next, we conducted a human clinical trial entitled, “Investigation of spinal cord regeneration therapy by cultured autologous bone marrow stromal cell transplantation for acute spinal cord injury, Phase I/II clinical trial.” No adverse events associated with cell transplantation were observed (Saito et al., 2008, 2012). However, the efficacy of bone marrow stromal cell infusion can obviously not be evaluated solely
on the basis of the results of 5 cases. To confirm the efficacy of the treatment, it is necessary to conduct clinical trials with a larger number of patients, and a low-cost and convenient method is required. However, bone marrow stromal cells must be cultured in a cell processing center for use, and the operation and maintenance of such a center incur a cost. This was found to be a bottleneck for a multicenter trial. Therefore, it was decided to conduct a clinical trial using autologous bone marrow mononuclear cells (BMMCs) that do not require cell culture manipulation, but are expected to have equal efficacy (Samdani et al., 2009). A phase I/II trial was conducted to assess the safety and feasibility after obtaining approval from our hospital’s Ethics Committee and the Committee on Guidelines for Stem Cell Research of the Ministry of Health, Labour and Welfare, Japan. As the patients participating in the study expected some positive effect, even if only a slight one, the Ethics Committee did not approve the establishment of a control group in which patients would have received normal saline solution injection instead of cells. 2. Materials and methods 2.1. Patient selection Patients who met all of the inclusion criteria and none of the exclusion criteria were considered eligible (Table 1). 2.2. Procedure for collection of bone marrow aspirate After local anesthesia was given, if needed, a syringe was used to collect bone marrow aspirate by inserting a bone marrow needle into the posterior iliac crest. In terms of the insertion sites on the skin, the needle was inserted into the ilium at two to three sites slightly away from each other. For each iliac insertion site, 4 mL of bone marrow aspirate was collected two to three times by changing the depth of insertion. From several sites on the left and right ilium, approximately 4 mL was collected each time; a total of approximately 120 mL was collected in the end. As bone marrow aspirate will turn into a gel if it is aspirated alone, 5 mL injection syringes, which had aspirated 1 mL of saline solution containing heparin beforehand, were used. Following this, mononuclear cells were isolated in an operating
Y. Suzuki et al. / Mononuclear cell transplantation for treatment of spinal cord injury
475
Table 1 Inclusion and exclusion criteria Inclusion criteria (1) Patients with spinal cord injuries classified as A–C on the ASIA impairment scale (2) Patients injured 3 weeks to 1 year previously (3) Patients with partial spinal cord injury demonstrated by diagnostic imaging (4) Patients aged between 20 and 60 years at the acquisition of informed consent (5) Patients who submitted written informed consent by themselves Exclusion criteria (1) Patients with a completely transected spinal cord (2) Patients with central spinal cord injury (3) Patients with organ failure with a SOFA score of 3 points or higher (4) Patients in whom hepatitis B, hepatitis C, human immunodeficiency virus infection, adult T-cell leukemia, or parvovirus B19 infection could not be ruled out (5) Patients with malignant tumor or a history of malignant tumor within 5 years (6) Patients with one of the following diseases/disorders: · Myeloproliferative disorder or myelodysplastic syndrome · Autoimmune disease · Spinal stenosis · Limb paralysis due to central nervous system disorder not attributed to spinal cord injury · Poorly controlled psychiatric disorder (7) Patients who were participating in other clinical trials or who completed participation within 6 months (8) Patients who were pregnant or possibly pregnant (9) Other patients who were judged to be ineligible by the investigators
Isolation, washing, and preparation of bone marrow mononuclear cells were performed on a clean bench in an operating room. The bone marrow aspirate was diluted with saline combined with human serum albumin and ACD-A solution (Terumo, Tokyo, Japan). Mononuclear cells were isolated by specific gravity centrifugation using Ficoll-Paque PREMIUM 1.073 (GE Healthcare, NY, USA). A part of the supernatant was used for a mycoplasma test, an endotoxin test, and a sterility test, and the rest was discarded. Cells were suspended in 2 mL of saline. Using some of the cells suspended in saline, hematological measurements, including cell count and cell surface markers, were performed. The survival rate of cells in the preliminary experiment was 99% or more when stored at room temperature for 12 hours; therefore, the cells were stored at room temperature until injection.
Japanese Pharmacopoeia, the notification by the Ministry of Health, Labor and Welfare titled “Ensuring the quality and safety of pharmaceuticals manufactured with components derived from human or animal as raw material” (Notification No. 1314 of the PFSB issued on Dec 26, 2000) and the minutes of the “Advisory Committee Regarding Clinical Trials Using Human Stem Cells, Council for Science and Technology, the Health Science Council,” and others. The mycoplasma test was performed using a PCR assay to examine the presence or absence of mycoplasma infection in the cells according to the 15th revised edition of Japanese Pharmacopoeia. The amount of endotoxin was measured in the cells according to the 16th revised edition of Japanese Pharmacopoeia. Measurement was performed using the supernatant removed after cell centrifugation in the procedure for the isolation of bone marrow mononuclear cells. Sterility test was performed by aerobic and anaerobic culture of the supernatant to examine the presence or absence of microorganism growth.
2.4. Quality control
2.5. Cell transplantation
For the purpose of quality control, the following tests were performed for the composition of the autologous bone marrow mononuclear cells for each subject. Quality control items were established with reference to the
The investigators confirmed that the level of endotoxin from the endotoxin test was
